DEVELOPMENTAL GENETICS 13:319-325 (1992)

REVIEW ARTICLE

The Genetic Program for Preimplantation Development GERALD M. KIDDER Department of Zoology, University of Western Ontario, London, Ontario N6A 5B7, Canada ABSTRACT This review summarizes information on accumulation profiles of individual gene transcripts in preimplantation development. Most of the information is from the mouse, but some data from other species are reviewed as well. The principal finding is that the transcription of most genes is not temporally linked with any of the three morphogenetic transitions (compaction, cavitation, and blastocoel expansion) that characterize this period. Most genes that are expressed during preimplantation development of the mouse are 01ready being transcribed in the 4-cell stage, and some clearly begin as early as the 2-cell stage. Once activated, a gene continues to be transcribed at least into the blastocyst stage, resulting in continuous rnRNA accumulation. Thus the pattern of gene transcription established at the time of genomic activation in the 2-cell stage is perpetuated into the blastocyst, with a few additions along the way. This information is interpreted in light of previous findings concerning the sensitivity of morphogenetic transitions to inhibition of gene expression. The lack of a clear relationship between the timing of expression of most genes and the schedule of morphogenesis leads one to conclude that temporal regulation is imposed downstream of transcription and translation. This conclusion is substantiated by a consideration of factors controlling the events of compaction. o 1992 WiIey-tiss, Inc. Key words: Mammalian embryos, compaction, cavitation, blastocoel expansion, gene transcription, mRNA

INTRODUCTION The period of mammalian development between fertilization and attachment to the uterine lining has received a great deal of attention in recent years, both because of the relative accessability of preimplantation embryos to experimentation, and because of their in-

0 1992 WILEY-LISS, INC.

creasing importance as a model for cellular morphogenesis and differentiation [Wiley et al., 19901. In addition, knowledge of physiological and molecular control mechanisms operating during this period is likely to have a n impact on reproductive technology. Embryos of eutherian mammals go through three major morphogenetic transitions leading up to implantation (Fig. 1). The first is known a s compaction, since its most obvious feature is the flattening of the blastomeres against one another until individual cell boundaries become indistinct. In the mouse this process occurs in the %cell stage and requires the Ca2+dependent cell-cell adhesion glycoprotein, E-cadherin [Shirayoshi et al., 1983; Johnson et al., 19881. Cell flattening is accompanied by blastomere polarization (each blastomere acquires apical and basolateral plasma membrane and cytocortical domains) and the assembly of specialized intercellular junctions, including gap and tight junctions [see Fleming and Johnson, 1988, for a review of compaction]. The second morphogenetic transition is cavitation, the process by which fluid accumulates between the blastomeres to form a blastocoel cavity. Cavitation depends on sodium pumps (N a+ ,K + ATPase) that become localized in the basolateral plasma membranes of the outer cell layer, the trophectoderm, and is facilitated by the expansion of focal tight junctions between trophectoderm cells [Watson and Kidder, 1988; Watson et al., 1990al. Finally, through the continued activity of sodium pumps, blastocoel expansion occurs. This is believed to facilitate hatching from the zona pellucida, the thick matrix surrounding the egg and embryo, enabling the embryo to

Received for publication June 16, 1992; accepted July 7, 1992. Address reprint requests to Dr. Gerald M. Kidder, Department of Zoology, The University of Western Ontario, London, Ontario N6A 5B7, Canada.

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0 d

a

- b

C

e

f

i

i

h

k

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Fig. 1. Morphogenetic transitions during mouse preimplantation development. The zona pellucida is not shown. The events of fertilization a-c are followed by the first cleavage (d),which occurs a t about 21 h. Genomic activation occurs during the 2-cell stage (d), which lasts about 18h. The 4-cell stage ( e )lasts about 1 2 h, as does the 8-cell stage (0.After the third cleavage, the process of compaction begins: blastomeres flatten against one another and become polarized, here indicated by the restriction of microvilli to the apical (outward-facing) poles (g). Specialized intercellular junctions also form during compaction. Differentiative divisions leading into the next cell cycle (16-cell

morula) (h) produce two distinct cell populations, a n outer, polar group and an inner, apolar group. When there are approximately 30 cells, cavitation begins with the accumulation of extracellular fluid between apposed blastomeres (i). Coalescence of this fluid gives rise to the blastocoel cavity of what is now known as a blastocyst (j). Further expansion of the blastocoel (k,l)is associated with rupture of the zona pellucida and hatching. At this point, the embryo is capable of initiating implantation. [From Wiley et al. (1990) with permission of The Company of Biologists Limited.]

come into direct contact with the uterus. The involvement of specific genes in these morphogenetic transitions has been reviewed recently [Kidder, 1993al.

mRNAs begin to decline, and is essentially complete by the time embryonic transcription has begun in the early cleavage stages [reviewed by Telford et al., 19901. An important consequence is that all phases of morphogenesis (if not all cleavages) prior to implantation are dependent on expression of embryonic genes. This is revealed by the existence of embryonic lethal mutations affecting preimplantation development [reviewed by Magnuson, 19861 and by the sensitivity of the three major morphogenetic transitions to agents which disrupt transcription or protein synthesis [Kidder and McLachlin, 1985; Levy et al., 1986; Seshagiri et al., 19921. Nonetheless, oogenetic gene products are likely to play important roles in preimplantation development since in the mouse there is evidence for the per-

THE OOGENETIC-EMBRYONIC TRANSITION Despite their dissimilarities, mammalian and nonmammalian zygotes employ the same basic strategy for the genetic programming of events immediately after fertilization: the stockpiling, in the growing oocyte, of gene transcripts that will be used later for the initiation of embryogenesis [reviewed by Schultz, 19861. In all embryos, this set of genetic instructions is eventually superseded by the products of embryonic transcription (the oogenetic-embryonic transition). In mammals, this transition starts with ovulation, as oogenetic

PREIMPLANTATION DEVELOPMENT sistence of some oogenetic mRNAs and proteins through the oogenetic-mbryonic transition [Taylor and Piko, 1987; Barron et al., 1989; Brenner et al., 1989; West and Flockhart, 19891. Most of the currently available information on the genetic program for preimplantation development comes from the mouse, although the body of data on the oogenetic-embryonic transition in other mammals is growing [Telford et al., 19901. It is clear that the timing of this transition is species dependent. It occurs in the 2-cell stage in mice, characterized by a precipitous decline in oogenetic mRNA, general activation of transcription, a corresponding shift in polypeptide synthesis pattern [documented especially clearly in the report by Latham et al., 19911, and the onset of sensitivity to the transcriptional inhibitor, a-amanitin. All but the first of these changes have been noted in human conceptuses during the fourth cell cycle (the 5- to 8-cell stage) [Braude et al., 1988; Grillo et al., 19911. Similar data indicate that the transition occurs in the 2-cell stage in hamsters [Seshagiri et al., 1990, 19921, in the 4-cell stage in pigs [Jarrell et al., 19911,and in the 8- to 16-cell stage of sheep and cattle [Telford et al., 19901. For cattle, however, this conclusion may have to be revised, since more recent evidence indicates that transcriptional activation actually occurs earlier [Barnes and First, 1991; L. Plante and W.A. King, personal communication]. In rabbits, the transition appears to be gradual, occurring between the 2- and 16-cell stages [Telford et al., 19901. The oogenetic-embryonic transition is most readily appreciated through examination of the accumulation profiles of individual mRNAs. One such study examined 37 unidentified rare to moderately abundant transcripts that were represented in a random cDNA library made from late 2-cell stage mouse RNA [Taylor and Pik6,1987]. Many of the 2-cell transcripts were not detected in ovulated oocytes and were therefore products of the embryonic genome, confirming that a major influx of new genetic information occurs in the cytoplasm of this species by the late 2-cell stage. Those transcripts that were oogenetic declined in abundance by about one-half, on average, between the 1- and 2-cell stages; most of them were subsequently replenished as the result of embryonic transcription. Nearly all of the transcripts accumulated continuously from the 2-cell stage onward, achieving a n average 15-fold increase in number of copies per embryo by the blastocyst stage. This observation implies that most genes, once activated in the 2-cell stage, continue to be transcribed throughout preimplantation development. This conclusion is substantiated by the accumulation profiles of a number of known mRNAs, summarized below.

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transcripts of known genes in mouse preimplantation development to provide a reasonably accurate view of the genetic program underlying morphogenesis and cell differentiation. This information is summarized in Table 1. The data are derived from those reports in which transcript presence was examined throughout preimplantation development, and where oogenetic transcripts had declined sufficiently by the 2-cell stage, so that embryonic transcripts could be unambiguously identified. A transcript is deemed to be accumulating (indicating transcription from the embryonic genome) when there is a quantitative increase in amount on a per embryo basis between two time points. Two fundamental conclusions emerge from these data. First, in the large majority of cases in which transcript level was examined in the 2- and 4-cell stages, accumulation of the mRNA was already occurring. Most of the known genes examined to date, then, begin transcription by the 4-cell stage, and possibly as early as the 2-cell stage. The second conclusion is that, once activated, a gene continues to be transcribed at least into the blastocyst stage resulting in continuous mRNA accumulation. Thus the pattern of gene transcription established by the late 2-cell stage is largely perpetuated into the blastocyst, with a few additions along the way. No mRNA has yet been identified which, once present, disappears a t a later stage of preimplantation development. This finding probably explains the relative constancy of polypeptide synthesis pattern from the late 2-cell stage onward [Levinson et al., 1978; Latham et al., 19911. What does this analysis tell us about the programming of individual morphogenetic transitions? Any attempt to construct a n hypothesis must take into account the known sensitivity of compaction, cavitation, and blastocoel expansion to transcriptional inhibitors. For compaction, the necessary transcriptional events are completed (or at least sufficient mRNAs have accumulated) by the mid-4-cell stage, and embryos are able to undergo compaction despite the presence of cxamanitin from that point onward [Kidder and McLachlin, 1985; McLachlin and Kidder, 1986; Levy et al., 19861. For cavitation, the coupling between transcription and morphogenesis is more immediate: mRNA required for the onset of fluid transport does not reach a sufficient level until the late morula stage [Kidder and McLachlin, 19851. Once the blastocoel has formed, development becomes less tightly coupled with transcription such that maximal expansion and escape from the zona pellucida can occur in blastocysts treated with a-amanitin for a t least 14 h [Kidder and McLachlin, 19851. A reasonable hypothesis, then, is that the genetic program for compaction in the mouse is established at the time of genomic activation in the 2-cell ACCUMULATION PROFILES OF stage, and the onset of compaction must therefore be KNOWN TRANSCRIPTS determined by later post-transcriptional events (furThere is now a sufficiently large body of information ther evidence in support of this view is considered in about the temporal pattern of accumulation of the the next section). The genetic program for cavitation,

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KIDDER TABLE 1. Accumulation Profiles of Known Transcripts in Preimplantation Development of the Mouse Begins to accumulate by 4-cell stage 8-cell stage' 8-cell stage'

Pattern Continuous Continuous Continuous

4- to 8-cell stage

Continuous

a-Tubulin E-Cadherin

4- to 8-cell stage 4-cell stage

Continuous Continuous

Connexin43

4-cell stage

Continuous

Northern blot, RT/PCR

Na',K+-ATPase al-subunit

4-cell stage

Continuous

Northern blot, RT/PCR

Na ,K+-ATPase pl-subunit E-, TN-type alkaline phosphatases GLUT-1 GLUT-2 TGF-a EGF-R TGF-Pl PDGF-A kFGF IGF-I1 IGF-11-R Insulin-R IGF-1-R CSF-1-R (c-fms) SF-R (c-kit) P-Glucuronidase HPRT Metallotheioneins 1, 2 Tissue-type plasminogen activator IAP-1. IAP-2 (retrovirus)

late morula

Continuous

2-cell stage

Continuous

Northern blot, RT/PCR RTIPCR

Morulaa 8-cell stage" 4-cell stage 8-cell stage 2-cell stage 4-cell stage 4-cell stageb 2-cell stage 2-cell stage 8-cell stage 8-cell stage 2-cell stage 2-cell stage 8-cell stage 4- to 8-stage-cell stage 2-cell stage 8-cell stagea

Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous Continuous

RT/PCR RT/PCR RT/PCR RTIPCR RT/PCR RT/PCR RT/PCR RT/PCR RT/PCR RT/PCR RT/PCR RTPCR RT/PCR Dot-blot Northern blot RT/PCR RT/PCR

8-cell stagea

Continuous

Slot-blot, RT/PCR

mRNA encoding Histone H3 Histones H2a,b Ribosomal proteins S15, L7a, L18a P, y-Actin

+

Method of detection Dot-blot Dot-b1ot Slot-blot Northern blot, slot-blot Northern blot Northern blot

References Graves et al., 1985 Graves et al., 1985 Taylor and Piko, 1991, 1992 Paynton et al., 1988; Taylor and Piko, 1990 Paynton et al., 1988 G.M. Kidder and 0. Blaschuk, unpublished data Valdimarsson et al., 1991; Nishi et al., 1991; Kidder, 1993b Watson et al., 1990b; Gardiner et al., 1990; Kidder, 1993b Watson et al., 1990b; Kidder, 1993b Hahnel et al., 1990 Hogan et al., 1991 Hogan et al., 1991 Werb, 1990 Wiley et al., 1992 Werb, 1990 Werb, 1990 Werb, 1990 Rappolee et al., 1992 Rappolee et al., 1992 Werb, 1990 Werb, 1990 Arceci et al., 1992 Arceci et al., 1992 Bevilacqua et al., 1988 Paynton et al., 1988 Andrews et al., 1991 X. Zhang and G.M. Kidder, unpublished data Poznanski and Calarco, 1991

ananscript presence was not examined in the 4-cell stage. bTranscript presence was not examined in the 2-cell stage.

on the other hand, must include one or a few genes whose transcripts begin to accumulate later in development in order to explain its greater sensitivity to a-amanitin. The gene encoding the Na+ ,K+-ATPase @l-subunitis a good candidate since its transcripts accumulate rapidly only after the morula stage, and the @-subunithas been implicated in regulating the expression of sodium pumps a t the plasma membrane [Watson et al., 1990b; Gardiner et al., 1990; Geering, 19911. The amazing a-amanitin insensitivity of blastocoel expansion and hatching implies that the gene expression pattern required for the onset of cavitation must be very stable, once established. Recent evidence obtained from bovine embryos suggests that the same principles probably apply in

the species a s well [Watson et al., 19921. RT/PCR was used to study the accumulation profiles of eleven different transcripts encoding a variety of growth factors and receptors. One (bFGF) is strictly oogenetic, since i t disappears by the time of genomic activation (8- to 16- cell stage) and does not reappear. The remaining oogenetic transcripts are superceded by embryonic transcripts, although this occurs later than in the mouse, after the 8-cell stage. Embryonic transcripts accumulate continuously from that stage onward. Only one mRNA, encoding bovine trophoblast protein (bTP), appeared for the first time in the blastocyst stage. Again, it appears that most of the genes involved in blastocyst formation are activated at once, at the time of genomic activation.

PREIMPLANTATION DEVELOPMENT

.-.. Cx43 ....

w

Cx43 mRNA nascent

b

Cx43 in gap junctions

time (hours)

.... 50

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55

Fig. 2. Summary of information concerning the regulation of gap junction assembly during compaction. The approximate time scale is given in hours post-fertilization. Transcripts encoding connexin43 (Cx43),a gap junction protein, begin to accumulate and are translated as early a s the 4-cell stage. Thus nascent Cx43 (indicated by the small dots) is present and increasing from the 4-cell stage onward. However, no Cx43 can be detected a t the blastomeres surfaces (and no gap

POST-TRANSCRIPTIONAL EVENTS UNDERLYING COMPACTION

60

rn

fi

65

...

70

junction are present) until after the onset of compaction, a t which time the connexin is incorporated into plasma membranes and into gap junctional plaques (indicated by solid bars within apposed membranes). This step is sensitive to the intracellular protein trafficking inhibitors, monensin and brefeldin-A. Gap junction assembly illustrates the importance of post-translational regulation in the temporal control of morphogenesis.

tion assembly cannot be either the transcription of the E-cadherin or connexin43 genes nor the translation of The two best-studied genes involved in compaction in their mRNAs. In the case of E-cadherin, the key event the mouse are those encoding the cell adhesion glyco- may be its redistribution from a global to a basolaterprotein, E-cadherin (also known a s uvomorulin), and ally restricted array in plasma membranes, possibly the gap junction channel-forming protein, connexin43. associated with its phosphorylation [Vestweber et al., The importance of E-cadherin for cell flattening was 1987; Sefton et al., 19921. Our analysis of the onset of mentioned earlier; connexin43 performs a n equally im- gap junctional coupling during compaction suggests portant function, a s i t contributes to the gap junctions that the regulated step in this case involves intracelthat form during compaction. Interference with either lular protein trafficking. of these proteins by means of antibodies or antisense Figure 2 summarizes our current view of the regunucleic acids results in the prevention of cell flattening lation of gap junction formation during compaction in or failure to maintain the compacted state [Shirayoshi the mouse. Experiments with cycloheximide, conet al., 1983; Johnson et al., 1986; Watson et al., 1990a; firmed by Western blotting, have demonstrated that Buehr et al., 1987; Lee et al., 1987; Bevilacqua et al., gap junction precursors (i.e. connexin43) are present a s 19891. Despite the involvement of both gene products early as the 4-cell stage, and intercellular coupling can in compaction, the onset of transcription in both cases be established a t the time of compaction using this limis a day earlier: both transcripts begin to accumulate a t ited precursor pool despite the virtual elimination of or before the 4-cell stage [Kidder and Blaschuk, unpub- protein synthesis [McLachlin et al., 1983; Valdimarslished Northern blot results with E-cadherin cDNA; son et al., 19911. Furthermore, the onset of gap juncValdimarsson et al., 1991; Nishi et al., 1991; Kidder, tional coupling is not linked to cell flattening itself, nor 1993133. Likewise, both proteins are already being syn- to the preceding rounds of cytokinesis or mitosis [Kidthesized by the 4-cell stage [Vestweber et al., 1987; der et al., 19871. The only agents able to interfere with Valdimarsson et al., 1991; Sefton et al., 1992; De Sousa, the establishment of intercellular coupling are those Valdimarsson, Nicholson, and G.M. Kidder, manu- such as monensin or brefeldin-A that block intracelluscript submitted]. Clearly, the developmentally regu- lar protein trafficking [De Sousa et al., manuscript sublated step in the onset of cell flattening and gap junc- mitted]. These agents disrupt the cytoplasmic arrays of

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nascent connexin43 that can be seen by confocal microscopy and prevent the connexin from entering plasma membranes but have no effect on established gap junctions. The fact that monensin or brefeldin-A can block the formation of gap junctions when applied a s late as the early (uncompacted) 8-cell stage suggests that the regulated step in this process is the transfer of nascent connexin43 from intracellular membranes to plasma membranes and thence into gap junction plaques. Experiments with somatic cell culture systems have implicated phosphorylation of nascent connexin43 as being associated with its assembly into gap junctions [Musil and Goodenough, 19911. Current investigation is directed a t determining what triggers the release of nascent connexin43 from its cytoplasmic location in precompaction embryos and how that event is linked with the developmental clock.

CONCLUSION The principal finding of this review is that the transcription of most genes in preimplantation development is not temporally linked with the morphogenetic transitions they participate in. The large majority of genes in the mouse whose mRNAs are present in the blastocyst are already being transcribed in the 4-cell stage. The few exceptions notwithstanding, this implies that post-transcriptional regulatory mechanisms must play a dominant role in determining the schedule of morphogenesis. In the case of gap junction formation, temporal regulation is linked to the insertion of nascent subunits into plasma membranes; their assembly into functional gap junction channels then follows quickly and perhaps spontaneously. While it is important to continue to identify genes that encode the building blocks for preimplantation morphogenesis, a real understanding of control mechanisms will require more research on the expression of genes involved in cellular regulation. ACKNOWLEDGMENTS The work from my laboratory reviewed in this article was supported by grants from the Natural Sciences and Engineering Research Council of Canada, the Medical Research Council of Canada, and the National Institutes of Health. The writing of this review was made possible by a Distinguished Research Professorship awarded by the Faculty of Science of the University of Western Ontario. REFERENCES Andrews GK, Huet-Hudson YM, Paria BC, McMaster MT, De SK, Dey SK (1991): Metallothionein gene expression and metal regulation during preimplantation mouse embryo development. Dev Biol 145:13-27. Arceci RJ, Pampfer S, Pollard JW (1992): Expression of CSF-lic-fms and SFic-kit mRNA during preimplantation mouse development. Dev Biol 151:l-8. Barnes FL. First NL (1991): Embryonic transcription in in vitro cultured bovine embryos. Mol Reprbd Dev 29:117-123.

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(1992):Golden hamster embryonic genome activation occurs a t the two-cell stage: Correlation with major developmental stages. Mol Reprod Dev 32:229-235. Shirayoshi Y, Okada TS, Takeichi M (1983): The calcium-dependent cell-cell adhesion system regulates inner cell mass formation and cell surface polarization in early mouse development. Cell 35631638. Taylor KD, Piko L (1987): Patterns of mRNA prevalence and expression of B1 and B2 transcripts in early mouse embryos. Development 101:877-892. Taylor KD, Piko L (1990): Quantitative changes in cytoskeletal p- and y-actin mRNAs and apparent absence of sarcomeric actin gene transcripts in early mouse embryos. Molec Reprod Dev 26:lll-121. Taylor KD, Piko L (1991):Expression of the rig gene in mouse oocytes and early embryos. Molec Reprod Dev 28:319-324. Taylor KD, Pik6 L (1992): Expression of ribosomal protein genes in mouse oocytes and early embryos. Mol Reprod Dev 31:182-188. Telford NA, Watson AJ, Schultz GA (1990): Transition from maternal to embryonic control in early mammalian development: A comparison of several species. Mol Reprod Dev 26:90-100. Valdimarsson G, DeSousa PA, Beyer EC, Paul DL, Kidder GM (1991): Zygotic expression of the connexin43 gene supplies subunits for gap junction assembly during mouse preimplantation development. Mol Reprod Dev 30:18-26. Vestweber D, Gossler A, Boller K, Kemler R (1987): Expression and distribution of cell adhesion molecule uvomorulin in mouse preimplantation embryos. Dev Biol 124:451-456. Watson AJ, Kidder GM (1988): Immunofluorescence assessment of the timing of appearance and cellular distribution of NaiK-ATPase during mouse embryogenesis. Dev Biol 126:80-90. Watson AJ, Damsky CH, Kidder GM (1990a): Differentiation of an epithelium: Factors affecting the polarized distribution of Na ,K ATPase in mouse trophectoderm. Dev Biol 141:104-114. Watson AJ, Pape C, Emanuel JR, Levenson R, Kidder GM (1990b): Expression of Na,K-ATPase a and p subunit genes during preimplantation development of the mouse. Dev Genet 11:41-48. Watson AJ, Hogan A, Hahnel A, Wiemer KE, Schultz GA (1992): Expression of growth factor ligand and receptor genes in the preimplantation bovine embryo. Mol Reprod Dev 31:87-95. Werb Z (1990): Expression of EGF and TGF-a genes in early mammalian development. Mol Reprod Dev 27:lO-15. West JD, Flockhart J H (1989): Genetic differences in glucose phosphate isomerase activity among mouse embryos. Development 107: 465-472. Wiley LM, Kidder GM, Watson AJ (1990): Cell polarity and development of the first epithelium. BioEssays 12:67-73. Wiley LM, Wu J-X, Harari I, Adamson ED (1992): Epidermal growth factor receptor mRNA and protein increase after the four-cell preimplantation stage in murine development. Dev Biol 149:247-260. +

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